Nucleic acid hybridization assays have proven useful in detecting the presence of microorganisms in biological samples (tissue, blood, urine, saliva, etc.) to diagnose infection and for detecting the presence in a mixture of a nucleic acid sequence of interest.
Nucleic acid hybridization assays are generally performed by immobilizing the test nucleic acid strand on a solid surface. A mixture containing the labeled complementary nucleic acid strand is contacted with the immobilized nucleic acid strand under conditions allowing hybridization of the two complementary strands. The hybridization of the two strands is then detected by chemical or other means by detecting the label on the probe strand. Alternatively, the nucleic acids are detected by sandwich hybridization techniques in which one set of sequences complementary to the target sequence is immobilized onto a solid surface. The surface is then contacted with the targer DNA and a labeled probe complementary to the target at a place different than the one attached to the solid surface. Under appropriate conditions the labeled probe is retained on the paper via the target DNA and detected by chemical or physical methods directly or indirectly.
Nucleic acids are typically immobilized on solid surfaces such as cellulose or nylon by physical contact with the surface, and the nucleic acids are bound to the surface through weak non-covalent bonds. Because of the nonspecific binding of nucleic acid to these surfaces, it is difficult to orient the nucleic acid so that it does not interact with the solid surface with the portions of the nucleic acid that are needed to hybridize with the test sequences. Thus greater amounts of the nucleic acid are needed to ensure a sufficient amount of free sequences to bind to the test nucleic acid. This inefficient use of nucleic acid can be expensive and can be limiting if only small quantities of the nucleic acid are available.
Additionally, the inability to accurately place the nucleic acid on the solid surface can hinder the effectiveness of the assay when small amounts of the nucleic acid sequence of interest are present, since it can be difficult to distinguish the nucleic acid present from background reactions.
Further, it would be desirable to be able to test for more than one pathogen in a single assay, or to test for more than one nucleic acid sequence in a single microorganism in the same assay. Uncertain placement of nucleic acids on the solid surface is a drawback to the development of assays of this kind.